A slow, tight binding inhibitor of InhA, the enoyl-acyl carrier protein reductase from Mycobacterium tuberculosis

Article abstract of DOI:10.1074/jbc.M109.090373

InhA, the enoyl-ACP reductase in Mycobacterium tuberculosis is an attractive target for the development of novel drugs against tuberculosis, a disease that kills more than two million people each year. InhA is the target of the current first line drug isoniazid for the treatment of tuberculosis infections. Compounds that directly target InhA and do not require activation by the mycobacterial catalase-peroxidase KatG are promising candidates for treating infections caused by isoniazid-resistant strains. Previously we reported the synthesis of several diphenyl ethers with nanomolar affinity for InhA. However, these compounds are rapid reversible inhibitors of the enzyme, and based on the knowledge that long drug target residence times are an important factor for in vivo drug activity, we set out to generate a slow onset inhibitor of InhA using structure-based drug design. 2-(o-Tolyloxy)-5- hexylphenol (PT70) is a slow, tight binding inhibitor of InhA with a K ₁ value of 22 pM. PT70 binds preferentially to the InhA·NAD⁺ complex and has a residence time of 24 min on the target, which is 14,000 times longer than that of the rapid reversible inhibitor from which it is derived. The 1.8 A crystal structure of the ternary complex between InhA, NAD⁺, and PT70 reveals the molecular details of enzyme-inhibitor recognition and supports the hypothesis that slow onset inhibition is coupled to ordering of an active site loop, which leads to the closure of the substrate-binding pocket.

Full text of DOI:10.1074/jbc.M109.090373

Protein Structure and Folding:
A Slow, Tight Binding Inhibitor of InhA,
the Enoyl-Acyl Carrier Protein Reductase
from Mycobacterium tuberculosis
Sylvia R. Luckner, Nina Liu, Christopher W.
am Ende, Peter J. Tonge and Caroline Kisker
J. Biol. Chem. 2010, 285:14330-14337.
doi: 10.1074/jbc.M109.090373 originally published online March 3, 2010
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 19, pp. 14330–14337, May 7, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
A Slow, Tight Binding Inhibitor of InhA, the Enoyl-Acyl Carrier
Protein Reductase from Mycobacterium tuberculosis*
Received for publication, December 1, 2009, and in revised form, February 14, 2010 Published, JBC Papers in Press, March 3, 2010, DOI 10.1074/jbc.M109.090373
Sylvia R. Luckner‡1, Nina Liu§1, Christopher W. am Ende§, Peter J. Tonge§2, and Caroline Kisker‡3
From the ‡Rudolf Virchow Center for Experimental Biomedicine, Institute for Structural Biology, University of Wu¨rzburg,
97080 Wu¨rzburg, Germany and the §Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook
University, Stony Brook, New York 11794-3400
InhA, the enoyl-ACP reductase in Mycobacterium tuberculo-
sis is an attractive target for the development of novel drugs
against tuberculosis, a disease that kills more than two million
people each year. InhA is the target of the current first line drug
isoniazid for the treatment of tuberculosis infections. Com-
pounds that directly target InhA and do not require activation
by the mycobacterial catalase-peroxidase KatG are promising
candidates for treating infections caused by isoniazid-resistant
strains. Previously we reported the synthesis of several diphenyl
ethers with nanomolar affinity for InhA. However, these com-
pounds are rapid reversible inhibitors of the enzyme, and based
on the knowledge that long drug target residence times are an
important factor for in vivo drug activity, we set out to generate
a slow onset inhibitor of InhA using structure-based drug
design. 2-(o-Tolyloxy)-5-hexylphenol (PT70) is a slow, tight
binding inhibitor of InhA with a K1 value of 22 pM. PT70 binds
months to nearly 2 years and thereby increasing the cost for
therapy 20-fold. Extensively drug-resistant strains, which are
almost untreatable with current chemotherapeutics, threaten
both developing and industrialized countries (2). Novel drugs
with activity against drug-resistant strains are therefore
urgently needed to restrain the disease that was once thought to
be under control.
One of the most effective and widely used drugs for the treat-
ment of tuberculosis is isoniazid (INH).4 INH is a prodrug that
has to be activated by KatG, the mycobacterial catalase-perox-
idase, to form together with NAD(H), an INH-NAD adduct (3).
This adduct is a slow onset inhibitor of InhA, the enoyl-ACP
reductase of the mycobacterial type II fatty acid biosynthesis
pathway (4, 5). In this pathway, very long chain fatty acids are
generated that act as precursors for mycolic acids, which in turn
are essential building blocks of the waxy cell wall of mycobac-
teria (6). Inhibition of InhA blocks mycolic acid biosynthesis,
thereby impairing the integrity of the cell wall and eventually
leading to cell death (7). Because the predominant mechanism
of resistance against isoniazid arises from mutations in KatG
(3), new compounds that directly target InhA and circumvent
the activation step are promising candidates for combating
multidrug-resistant strains of M. tuberculosis.
؉
preferentially to the InhA⅐NAD complex and has a residence
time of 24 min on the target, which is 14,000 times longer than
that of the rapid reversible inhibitor from which it is derived.
˚
The 1.8 A crystal structure of the ternary complex between
؉
InhA, NAD , and PT70 reveals the molecular details of enzyme-
inhibitor recognition and supports the hypothesis that slow
onset inhibition is coupled to ordering of an active site loop,
which leads to the closure of the substrate-binding pocket.
Triclosan inhibits InhA directly (8). Although it is a relatively
weak inhibitor of mycobacterial InhA (Ki ϭ 0.2 ␮M (9)), signif-
icant progress has been made to improve its affinity toward
InhA; first generation diphenyl ethers have been developed
using structure-based drug design that are nanomolar inhibi-
tors of InhA with minimum inhibitory concentrations of 1–2
␮g/ml against both drug-sensitive and drug-resistant strains of
M. tuberculosis (10). In addition, Freundlich et al. (11) recently
reported the development of potent triclosan derivatives that
demonstrated inhibition of InhA in the nanomolar range with
minimum inhibitory concentrations of 5–10 ␮g/ml. However,
all compounds in Ref. 10 are rapid reversible inhibitors of the
enzyme. This is significant given the increasing importance
attached to compounds that have long residence times on their
targets (see Ref. 12 and references therein), as recently demon-
strated by the correlation between residence time and in vivo
activity for a series of Francisella tularensis FabI inhibitors (13).
In addition, the highly successful INH-NAD adduct was shown
Mycobacterium tuberculosis is the causative agent of tuber-
culosis, an infectious disease that is spread all over the world.
Two billion people, one-third of the world population, are
infected with tuberculosis, and M. tuberculosis is responsible
for 8.8 million new infections and 1.6 million deaths each year
(1). The emergence of multidrug-resistant M. tuberculosis
strains that are resistant against the current frontline drugs iso-
niazid and rifampicin, contribute to the spread and worsen the
situation by lengthening the treatment considerably from 6
* Thisworkwassupported, inwholeorinpart, byNationalInstitutesofHealth
Grant AI44639 (to P. J. T.). This work was also supported by Deutsche
Forschungsgemeinschaft Grant SFB 630 and Forschungszentrum Grant
FZ82 (to C. K.).
The atomic coordinates and structure factors (codes 2x22 and 2x23) have been
deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, NewBrunswick, NJ(http://www.rcsb.org/).
1 Both authors contributed equally to this work.
2 To whom correspondence may be addressed. Tel.: 631-632-7907; Fax: 631- 4 The abbreviations used are: INH, isoniazid; 6PP, 5-hexyl-2-phenoxyphenol;
632-7960; E-mail: peter.tonge@sunysb.edu.
8PP, 5-octyl-2-phenoxyphenol; ACP, acyl carrier protein; DD-CoA, trans
2-dodecenyl-CoA; PT70, 2-(o-tolyloxy)-5-hexylphenol; PIPES, 1,4-pipera-
zinediethanesulfonic acid; DMSO, dimethyl sulfoxide.
3 To whom correspondence may be addressed. Tel.: 49-931-31-80381; Fax:
49-931-31-87320; E-mail: caroline.kisker@virchow.uni-wuerzburg.de.
14330 JOURNAL OF BIOLOGICAL CHEMISTRY
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Slow Onset Inhibition of InhA
M solution in tetrahydrofuran; 4.0 ml, 4.26 mmol) was then
added dropwise, and the resulting solution was stirred at room
temperature for 20 min. N-Methylpyrrolidinone (4.7 ml) was
then added to the flask, followed by 21.7 mg (0.0426 mmol) of
Pd(P(t-Bu)3)2 and 500 mg (1.71 mmol) of 1-(4-bromo-2-me-
thoxyphenoxy)-2-methylbenzene after 5 min. The flask was fit-
ted with a reflux condenser and heated for 48 h at 130 °C. After
cooling gradually to room temperature, 20 ml of a 1.0 M aque-
ous HCl solution was added to the flask. The resulting mixture
was extracted with diethyl ether, and the ether extract was
washed with water, dried over anhydrous MgSO4, and concen-
trated under vacuum to give the crude product 1-(4-hexyl-2-
methoxyphenoxy)-2-methylbenzene, which was purified by
flash column chromatography on silica gel (ethyl acetate/hex-
ane). A solution of boron tribromide in 1.8 ml of dichlorometh-
ane (1.0 M solution) was added to a solution of the protected
diphenyl ether (1.4 mmol) in 3 ml of dry dichloromethane at
Ϫ78 °C for 1 h and then at room temperature for 3 h. When thin
layer chromatography showed that the reaction had reached
completion, the reaction mixture was quenched with methanol
at Ϫ78 °C and concentrated to give an oil. A suspension of this
oil in dichloromethane was washed with 10% aqueous sodium
bicarbonate solution, and the organic layer was drawn off,
washed sequentially with water then brine, and dried over
MgSO4. The organic layer was filtered and concentrated under
vacuum to give the crude product, which was then purified by
flash column chromatography (ethyl acetate/hexane). 1H NMR
(300 MHz, CDCl3): ␦ 0.87–0.92 (m, 3H), 1.27–1.36 (m, 6H),
1.57–1.63 (m, 2H), 2.30 (s, 3H), 2.52–2.57 (t, J ϭ 7.5 Hz, 2H),
5.56 (s, 1H), 6.61–7.26 (m, 7H); ESI-MS (m/z): 285.1 (Mϩ1).
Substrate Preparation—Trans-2-dodecenoyl-CoA (DD-CoA)
was synthesized from trans-2-dodecenoic acid by using the
mixed anhydride method as described previously (17). Ali-
quots of the substrate were flash frozen in liquid N2 and
stored at Ϫ80 °C.
FIGURE 1. Structures of the rapid reversible inhibitor 6PP and the slow
onset inhibitor PT70.
to be a slow onset inhibitor of InhA (5). The crystal structures of
InhA (Protein Data Bank code 2nv6 (14)) and Escherichia coli
FabI (Protein Data Bank code 1qg6 (15)) support the hypothesis
that slow onset inhibition is coupled to ordering of an active site
loop (residues 195–210 in InhA), which leads to a closure of the
substrate-binding pocket.
Toward the design of a slow onset diphenyl ether, we specu-
lated that there must be an entropic penalty for loop ordering.
Thus, reducing the conformational flexibility of the lead
diphenyl ether might enable ordering of the active site loop,
thus in turn resulting in slow onset enzyme inhibition. Intro-
duction of a methyl group ortho to the diphenyl ether linkage
resulted in a compound, PT70 (Fig. 1), that is a slow onset inhib-
itor of InhA with a K1 value of 22 pM. The crystal structure of
PT70 bound to InhA corroborates that slow onset inhibition is
coupled to ordering of the substrate-binding loop. Significantly
the B-ring methyl group of PT70 forms critical van der Waal’s
ϩ
interactions with the NAD cofactor and with key amino acids
of the formerly disordered substrate-binding loop. These stud-
ies provide information on the mechanistic imperatives for
slow onset inhibition of enoyl ACP reductases, and the novel
inhibitor has the potential to be effective against both drug-
sensitive and drug-resistant strains of M. tuberculosis.
Expression and Purification of InhA—InhA, the M. tubercu-
losis enoyl-ACP reductase, was expressed in E. coli strain
EXPERIMENTAL PROCEDURES
Synthesis of PT70—PT70 was synthesized using the proce- BL21(DE3)pLysS. The cells were induced with 1 mM isopropyl-
dure described previously for the synthesis of alkyl-substituted ␤-D-thiogalactopyranoside at an A600 of 0.8 and harvested by
diphenyl ethers (16). Briefly, 2-iodotoluene (7.35 mmol), 4-bro- centrifugation after shaking for 12 h at 25 °C. The bacterial
mo-2-methoxyphenol (14.7 mmol), Cs2CO3 (32.3 mmol), pellet was resuspended in 20 mM Tris-HCl, pH 7.9, containing
(CuOTf)2⅐PhH (0.735 mmol, 5.0 mol % copper), ethyl acetate 500 mM NaCl and 5 mM imidazole and lysed by sonication fol-
(0.125 mmol, 5.0 mol %), 1-naphthoic acid (32.3 mmol), molec- lowed by centrifugation at 50,000 ϫ g to remove cell debris. The
ular sieves 4 Å (1.8 g), and toluene (15 ml) were added to an supernatant was applied to a nickel affinity column (GE Health-
oven-dried 50-ml two-necked round-bottomed flask sealed care), and InhA was eluted using a gradient of 60–500 mM
with a septum that was purged with N2 and heated to 110 °C. imidazole. Fractions containing InhA were pooled and
After 24 h the reaction mixture was cooled to room tempera- exchanged into 30 mM PIPES buffer, pH 6.8, containing 150 mM
ture, dichloromethane was added, and the organic phase was NaCl and 1 mM EDTA via desalting columns (GE Healthcare).
obtained by filtration. The organic phase was washed with 5% For further purification, size exclusion chromatography was
NaOH, and the aqueous layer was then extracted with dichlo- carried out using a Superdex 200 column (GE Healthcare) and
romethane. The combined organic layers were washed with the same buffer.
brine, dried over MgSO4, and concentrated under vacuum to
Steady State Kinetic Assay—Kinetic assays using DD-CoA
give the crude product (1-(4-bromo-2-methoxyphenoxy)-2- and wild-type InhA were performed as described previously (9).
methylbenzene), which was subsequently purified by flash col- The reactions were initiated by the addition of InhA to solu-
umn chromatography on silica gel (ethyl acetate/hexane). tions containing substrate, inhibitor, and NADH in 30 mM
ZnCl2 (0.5 M solution in tetrahydrofuran; 9.0 ml, 4.52 mmol) PIPES and 150 mM NaCl, pH 6.8. The IC50 values were deter-
was added to a round-bottomed flask sealed with a rubber sep- mined by varying the concentration of inhibitor in reactions
tum and purged with nitrogen. Hexyl magnesium chloride (1.0 containing 250 ␮M NADH, 25 ␮M DD-CoA, and 100 nM InhA.
MAY 7, 2010•VOLUME 285•NUMBER 19
JOURNAL OF BIOLOGICAL CHEMISTRY 14331

Slow Onset Inhibition of InhA
When the assays were performed at 10 nM InhA, the enzyme tion was initiated by the addition of DD-CoA (30 ␮M). Equation
was stabilized by the addition of glycerol (8%, v/v) and bovine 5 was used to estimate the apparent inhibition constant KiЈ,
serum albumin (0.1 mg/ml). The experimental data were ana-
v ϭ v0/͑1 ϩ ͓I͔/KiЈ͒
(Eq. 5)
lyzed using Equation 1, where I is the inhibitor concentration,
and y is the percentage of activity.
where v and v0 are the initial velocity in the presence and
absence of inhibitor, respectively, and [I] is the inhibitor con-
y ϭ 100%/͓1 ϩ ͑I/IC50͔͒
(Eq. 1)
ϩ
centration. The KiЈ values obtained at different NAD concen-
trations were then fit to Equations 6–8, which describe the
Data fitting was performed using Grafit 4.0 (Erithacus Software
Ltd.).
ϩ
binding of the inhibitor to either E-NAD (Equation 6),
ϩ
E-NADH (Equation 7), or both E-NAD and E-NADH (Equa-
Progress Curve Analysis—The slow onset inhibition of InhA
by PT70 was monitored by adding the enzyme (5 nM) to assay
mixtures containing glycerol (8%), bovine serum albumin (0.1
mg/ml), DMSO (2% v/v), DD-CoA (300 ␮M), NADH (250 ␮M),
tion 8).
KiЈ ϭ K1͑1 ϩ Km NAD/͓NADϩ͔͒
KiЈ ϭ K2͑1 ϩ ͓NADϩ͔/Km NAD
(Eq. 6)
(Eq. 7)
ϩ
NAD (200 ␮M), and inhibitor (0–480 nM). The reactions were
͒
monitored until the progress curve became linear, indicating
that the steady state had been reached. To ensure that substrate
depletion would not significantly affect the reaction rate, low
enzyme concentrations and high substrate concentrations were
used. Progress curves were analyzed as described previously
(13, 18). This involved fitting the data to Equation 2,
KiЈ ϭ K2͑1 ϩ ͓NADϩ͔/Km NAD͒/͓1 ϩ ͓NADϩ͔/͑Km NADK1/K2͔͒
(Eq. 8)
K1 and K2 are inhibition constants for inhibitor binding to the
ϩ
E-NAD and E-NADH forms.
Direct Determination of koff—Having shown that PT70
At ϭ A0 Ϫ vst Ϫ ͑vi Ϫ vs͒͑1 Ϫ ␥͒*ln͕͓1
ϩ
bound preferentially to the E-NAD product complex, the rate
of dissociation of PT70 from InhA was monitored using 32P-
Ϫ ␥*exp͑Ϫkobst*t͔͒/͑1 Ϫ ␥͖͒/͑kobs*␥͒ (Eq. 2)
ϩ
NAD to provide a direct estimate for koff. These experiments
where ␥ ϭ [E]*(1 Ϫ vs/vi)2/[I], vi and vs are the initial velocity
and steady state velocity, and kobs is the observed rate constant
for each progress curve. The values of kobs, vi, and vs obtained
from Equation 2 were then fitted to Equations 3 and 4, which
describe a two-step inhibition mechanism in which rapid bind-
ing of the inhibitor to the enzyme is followed by a second slow
step that results in the final complex.
followed a similar protocol to that described previously (5).
ϩ
ϩ
InhA was incubated with PT70, NAD , and 32P-NAD (800
Ci/mmol) for 5 h at room temperature to generate the ternary
ϩ
complex formed by InhA, NAD , and PT70. After purification
using a Sephadex G-75 spin column, 500 ␮l of the complex was
diluted into 70 ml of buffer to initiate the dissociation of the
ϩ
inhibitor from the enzyme. Because NAD only has a weak
affinity for the free enzyme (Kd Ͼ 2 mM), dissociation of PT70
k
obs ϭ kϪ2 ϩ k2͓I͔/͑KaϪp1p ϩ [I]͒
(Eq. 3)
ϩ
from the ternary complex also leads to the release of NAD and
32P-NAD from the enzyme. Subsequently, 700 ␮l of the
ϩ
vs/v0 ϭ 1/͑1 ϩ ͓I͔/Kiapp
͒
(Eq. 4)
diluted complex solution was withdrawn at various time inter-
vals, loaded into a microcon (Satorious 500, 10 kDa), and cen-
trifuged in a microcentrifuge for 1 min at maximum speed. 450
In these equations, Kapp and Kai pp are the apparent dissociation
constants for the initiϪal1enzyme⅐inhibitor complex (E-I) and the
final enzyme⅐inhibitor complex (E-I*), respectively.
␮l of the filtrate was collected, and the amount of 32P-NAD
ϩ
was quantitated using a scintillation counter. A value for koff
was obtained by fitting the data to Equation 9, where Nt is the
number of radioactive counts (cpm) at time t, and N0 is the
number of radioactive counts (cpm) following complete disso-
ciation of the complex.
In addition to monitoring the onset of enzyme inhibition,
progress curves were also used to analyze the recovery of
enzyme activity resulting from the slow dissociation of inhibi-
tor from E-I*. InhA (0.5 ␮M) was preincubated with PT70 (0.3–
ϩ
0.9 ␮M) and NAD (200 ␮M) at room temperature. After 5 h, 5
␮l of incubation mixture was diluted 100-fold into an assay
Nt ϭ N0͑1 Ϫ exp͑Ϫkofft͒͒
(Eq. 9)
mixture containing glycerol (8%), bovine serum albumin (0.1
mg/ml), DMSO (2% v/v), DD-CoA (300 ␮M), NADH (250 ␮M),
Crystallization—For the formation of the ternary InhA⅐
ϩ
ϩ
and NAD (200 ␮M). Substrate consumption was monitored at NAD ⅐PT70 complex, the protein solution (10 mg/ml) was
ϩ
340 nm, and the resulting recovery progress curves were ana- combined with NAD and PT70 in a molar ratio of 1:5:200.
lyzed in a similar fashion to that described above (18, 19).
After 2 h of incubation on ice, the solution was centrifuged at
Preincubation Inhibition Assays for Slow Binding Inhibitors— 25,000 ϫ g for 20 min, and the supernatant was used for crys-
Preincubation inhibition assays were performed to determine tallization by the hanging drop vapor diffusion technique. Equal
the preference of PT70 for the different cofactor-bound forms volumes of protein solution and precipitant solution (12–16%
of InhA. These experiments were conducted as described pre- (w/v) polyethylene glycol 4000, 1% DMSO, 100 mM N-(2-acet-
viously (18). InhA (10 nM) was preincubated in the presence of amido) iminodiacetic acid, pH 6.8, 100–250 mM ammonium
ϩ
a fixed concentration of DMSO (2%), NAD (10–200 ␮M), acetate) were mixed and equilibrated against precipitant solu-
NADH (250 ␮M), and PT70 (0–1000 ␮M) for 5 h at 4 °C. The tion. Diffraction quality crystals of the complex grew within 4
mixture was then warmed to room temperature, and the reac- days at 22 °C to a maximum size of 900 ϫ 100 ϫ 100 ␮m3. The
14332 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 19•MAY 7, 2010

Slow Onset Inhibition of InhA
unambiguous modeling of the
ϩ
NAD
molecule, the inhibitor
PT70, and the missing residues of
the substrate-binding loop into the
electron density with Coot.
For data set 2, the coordinates of
one monomer of the refined first
structure, omitting only the inhibi-
tor, were used as a search model.
Molecular replacement yielded one
homotetramer in the crystallo-
graphic asymmetric unit. Refine-
ment and model building was car-
ried out as described above. The
refinement statistics for the two
data sets are listed in Table 2. The
Ramachandran statistics calculated
by MolProbity (25) show that the
structures are of good quality. In
structure 1, 96.9% of all residues are
in the favored regions, and 100% are
in the allowed regions. 96.4% of all
residues of structure 2 are in the
favored regions, and 99.9% in the
allowed regions. Residue Asp42,
located in a surface exposed loop, is
an outlier of the Ramachandran
plot. All of the structural figures
were prepared with Pymol (26).
FIGURE 2. Progress curve analysis for the inhibition of InhA by PT70 and effect of NAD؉ on the apparent
inhibition constant of PT70. A, progress curves were obtained for inhibitor concentrations ranging from 0 to
480 nM. The solid curves are the best fits of the data to Equation 2. B, kobs plotted as a hyperbolic function of
[PT70] using Equation 3. C, progress curves of InhA activity recovery were obtained for inhibitor concentrations
ranging from 0 to 9 nM. The solid curves are the best fits of the data to Equation 2 to obtain kobs, which was then
plotted against [PT70] using Equation 3 to obtain kϪ2. D, effect of NADϩ on the apparent inhibition constant of
PT70. The fitted curves are shown for Equation 6 (dashed line; K1 ϭ 0.0146 Ϯ 0.0009 nM), Equation 7 (dotted line;
K2 ϭ 13.6 Ϯ 5.5 nM), and Equation 8 (solid line; K1 ϭ 0.022 Ϯ 0.001 nM, K2 ϭ 90.6 Ϯ 9.7 nM).
RESULTS
Kinetic Characterization—The inhibition of InhA by PT70
was characterized by determining the IC50 values using steady
state kinetic methods. The IC50 values obtained using 100 nM
FIGURE 3. Kinetic scheme for the slow onset inhibition of InhA by PT70.
crystals were transferred to precipitant solutions containing and 10 nM InhA were 50.3 Ϯ 7.0 and 5.3 Ϯ 0.4 nM, respectively,
ϩ
NAD and PT70 in excess and increasing amounts of DMSO in indicating that PT70 is a tight binding inhibitor. Progress curve
steps of 5% (v/v) to a final concentration of 25% and subse- analysis was used to determine whether PT70 is also a slow
quently frozen in liquid nitrogen.
onset inhibitor of InhA. The reaction conditions were adjusted
Data Collection, Structure Determination, and Refinement— so that consumption of DD-CoA occurred at a linear rate for up
Two diffraction data sets were collected at a Rigaku Micro- to 30 min. Upon addition of the inhibitor, the turnover velocity
MaxTM-007HF x-ray generator with an Raxis HTC detector. decreased exponentially with time, from an initial velocity (vi)
The first crystal diffracted to 2.1 Å, and the second crystal dif- to a steady state velocity (vs). Additionally, with increasing con-
fracted to 1.8 Å. Data sets 1 and 2 were indexed and processed centrations of PT70, both vi and vs decreased, whereas kobs
using Mosflm (20) and XDS (21), respectively. Although the increased, and the time required to reach vs decreased (Fig. 2A).
crystals grew in the same drop, they belong to different space This behavior is a classic example of slow onset inhibition in
groups: C2221 for data set 1 and P21 for data set 2. The data which the rapid formation of the initial E-I complex is followed
collection statistics are listed in Table 2. The two structures by a second slow step leading to the formation of the final E-I*
were solved by molecular replacement using Phaser (22). As a complex (Fig. 3). Fitting the data to Equation 2 provided values
search model for data set 1, a monomer was derived from the for vi, vs, and kobs. The hyperbolic dependence of kobs on the
ϩ
Protein Data Bank entry 2b37 (10), omitting the NAD cofac- concentration of PT70 was fitted to Equation 3, allowing the
tor and the inhibitor. Molecular replacement yielded two mol- calculation of the constants for the conversion of E-I to E-I* (k2
ecules in the crystallographic asymmetric unit that form half of and kϪ2 in Fig. 3) and also providing a value for KaϪp1p, the disso-
a homotetramer. Model building and refinement were carried ciation constant of E-I (Fig. 2B). The dependence of vs/v0 on the
out using alternating rounds of Coot (23) for manual model concentration of PT70 was analyzed using Equation 4 to give
building and Refmac (24) for maximum likelihood refinement. Kai pp, the dissociation constant of the final E-I* complex. The
Five percent of all reflections were omitted throughout the thermodynamic and kinetic constants describing the inhibition
refinement for the calculation of Rfree. In the initial refinement of InhA by PT70 are summarized in Table 1. PT70 initially
app
rounds, the density improved significantly and allowed the binds to InhA with a K value of 62 nM, whereas the dissoci-
Ϫ1
MAY 7, 2010•VOLUME 285•NUMBER 19
JOURNAL OF BIOLOGICAL CHEMISTRY 14333

Slow Onset Inhibition of InhA
TABLE 1
Inhibition of InhA by PT70 and the INH-NAD adduct
2a
app
b
c
Inhibitor
k2
min
0.46 Ϯ 0.003
0.46 Ϯ 0.003
0.13 Ϯ 0.01
k؊2
min
t1⁄
Residence timea
K
Kai pp
nM
7.8 Ϯ 0.4
7.8 Ϯ 0.4
5.0 Ϯ 0.5
koff
min
K1
؊1
Ϫ1
Ϫ1
Ϫ1
min
min
nM
pM
PT70
0.041 Ϯ 0.003e
0.044 Ϯ 0.008f
0.016 Ϯ 0.007
17 Ϯ 1
17 Ϯ 1
43 Ϯ 12
24 Ϯ 2
24 Ϯ 2
63 Ϯ 27
62 Ϯ 2
0.043 Ϯ 0.006
0.043 Ϯ 0.006
0.017 Ϯ 0.001
22 Ϯ 1
PT70
62 Ϯ 2
22 Ϯ 1
INH-NADd
100 Ϯ 75
a t ⁄
1
2 ϭ 0.693/koff and residence time ϭ 1/koff, where koff ϭ kϪ1kϪ2/(kϪ1 ϩ k2 ϩ kϪ2). Assuming that kϪ1 ϾϾ k2 and kϪ2, then koff Ϸ kϪ2
.
b koff was determined by monitoring the rate of release of 32P-NAD from the ternary enzyme⅐inhibitor complex.
c K1 was determined from preincubation experiments.
ϩ
d The data were taken from Ref. 5.
e kϪ2 was determined by progress curve of enzyme⅐inhibitor complex formation.
f kϪ2 was determined by progress curve of enzyme activity recovery.
ation constant of the final E-I* complex Kai pp is ϳ10 times
TABLE 2
app
Crystallographic data and refinement statistics
smaller than K (7.8 nM). Data analysis using Equations 2 and
Ϫ1
؉
InhA NAD PT70
Crystal 1 Crystal 2
3 also provided a value for kϪ2, the rate constant for conversion
of E-I* to E-I. Assuming that kϪ1 ϾϾ k2 and kϪ2, then kϪ2 will be
equal to koff, the dissociation rate constant for the formation of
active enzyme from E-I*. The value of kϪ2 obtained from pro-
gress curve analysis of 0.041 minϪ1 is the same within experi-
mental error to the values obtained when the recovery of
enzyme activity was monitored (Fig. 2C; 0.044 minϪ1) and
when koff was determined directly by following the dissociation
Data collection
Space group
Cell dimensions
a, b, c (Å)
C2221
P21
89.80, 157.51, 91.23
90.00, 90.00, 90.00
88.48, 90.27, 89.56
90.00, 118.76, 90.00
␣, ␤, ␥ (°)
Resolution (Å)
Rmerge
36.16-2.10 (2.21-2.10) 78.57-1.81 (1.9-1.81)
0.080 (0.300)
16.0 (2.5)
98.1 (89.7)
4.4 (2.9)
0.101 (0.438)
6.1 (1.7)
I/␴ I
Completeness (%)
92.5 (77.8)
1.6 (1.4)
Redundancy
ϩ
of 32P-NAD from the enzyme⅐inhibitor ternary complex
Refinement
(0.043 minϪ1). Thus, the residence time of PT70 on the enzyme
(1/koff) is 24 min, which is ϳ3-fold smaller than that of the
INH-NAD adduct (Table 1). Most diphenyl ethers preferen-
Resolution (Å)
No. reflections
2.10
38141
1.81
112861
16.8/20.3
R
work/Rfree (%)
17.2/21.6
No. atoms
Protein
PT70
4063
42
8033
84
ϩ
tially bind to the E-NAD product complex of InhA and other
ϩ
NAD
88
176
682
FabI enzymes and only occasionally prefer the E-NADH form
of the enzyme (9, 10, 13, 27–29). Preincubation studies were
used to examine which form of the enzyme PT70 prefers. InhA
and PT70 were preincubated in the presence of saturating
Water
B-factors
Protein
PT70
143
26.2
26.9
19.7
29.9
10.4
7.3
ϩ
NAD
3.9
ϩ
Water
16.0
NADH (250 ␮M) and at different fixed concentrations of NAD
Root mean square deviations
Bond lengths (Å)
Bond angles (°)
0.013
1.465
0.015
1.534
(10, 30, 50, 100, and 200 ␮M). Assays were initiated by adding
the substrate DD-CoA to obtain the apparent inhibition con-
ϩ
stant KiЈ at each concentration of NAD . The dependence of
ϩ
KiЈ on the concentration of NAD was fit to Equations 6–8
rable with the rings of the diphenyl ethers 5-pentyl-2-phenoxy-
phenol and 8PP described previously (10). Hydrogen bonds are
formed between the inhibitor hydroxyl group and Tyr158, and a
hydrogen bonding network is formed between the 2Ј-hydroxyl
using a Km NAD value of 4 mM and a Km NADH value of 48 ␮M.
ϩ
The dependence of KiЈ on the concentration of NAD was best
described by Equation 8 (Fig. 2D), indicating that PT70 binds to
ϩ
both the E-NAD and E-NADH forms of the enzyme, albeit
group of NAD and Lys165 (Fig. 5, red dotted lines). A ␲-␲
ϩ
with a strong (4000-fold) preference for the enzyme-oxidized
cofactor product complex (K1 ϭ 0.022 nM, K2 ϭ 90.6 nM; Table
1). The value of K1 is significantly smaller than that obtained for
Kai pp (7.8 nM); however, the latter was determined at a fixed
stacking interaction between the B-ring of PT70 and the nico-
ϩ
tinamide ring of NAD further stabilizes the conformation of
the inhibitor. The alkyl chain of PT70 extends into the hydro-
phobic environment of the substrate binding cavity and forms
ϩ
concentration of NAD .
hydrophobic interactions with residues Phe149 and Tyr158
.
ϩ
Structure of the Ternary InhA⅐PT70⅐NAD Complex—The
Most importantly, however, is the substrate-binding loop (res-
idues 195–210, helix ␣6) of the structures reported here, which
is ordered and forms a helix that covers the entrance to the
active site (Fig. 4). Hydrophobic interactions are formed
between the phenyl rings of the inhibitor and amino acids
Ala198, Ile202, Val203, and Met199 of the substrate-binding loop
(Fig. 5). Ala198 forms hydrophobic interactions with the B-ring
methyl group at a distance of 3.4 Å, Ile202 interacts with the
B-ring at a distance of 3.7 Å, and Met199 interacts with the
A-ring at a distance of 3.8 Å. Val203 forms hydrophobic inter-
actions with both phenyl rings at a distance of 4 Å and with the
binding of PT70 to InhA and the basis for the slow binding step
was further characterized by structural studies. Two structures
ϩ
of the ternary InhA⅐NAD ⅐PT70 complex were solved. The
first structure was solved at a resolution of 2.1 Å resolution and
belongs to space group C2221, the same space group as in the
ternary InhA complex with 5-octyl-2-phenoxyphenol and
ϩ
NAD (8PP; Protein Data Bank code 2b37 (10)). The second
structure was obtained at 1.8 Å resolution and belongs to space
group P21, which has not been reported for InhA so far (struc-
tural data are summarized in Table 2) (Fig. 4). The slow onset
inhibitor PT70 binds to the substrate-binding site with the two
rings of the inhibitor oriented almost 90° to each other, compa- acyl chain of PT70 at 3.7 Å.
14334 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 19•MAY 7, 2010

Slow Onset Inhibition of InhA
FIGURE 5. Close-up of the binding pocket of InhA with bound NAD؉ and
PT70. HydrogenbondsbetweenPT70(black)andTyr158 (yellow)aswellasthe
NADϩ molecule (cyan) are indicated as red dotted lines. The important hydro-
phobicresiduesAla198, Met199, Ile202, andVal203 ofthesubstrate-bindingloop
are shown in all bonds representation (red). An overlay of different triclosan
inhibitors (PT70, black; triclosan, gray; 8PP, blue; JPL, green) in the binding
pocket of InhA displays the differences in the orientation of the B-ring.
results, we rationally modified the alkyl-diphenyl ethers to pro-
mote interactions between the inhibitor and the loop that
becomes ordered during slow onset inhibition.
FIGURE 4. Loop ordering upon slow binding inhibition. A, one monomer of
the ternary InhA⅐NADϩ⅐8PP complex (Protein Data Bank code 2b37) is shown
in cartoon representation with the NADϩ molecule in cyan and the 8PP mol-
ecule in black and all-bonds representation. The substrate-binding loop is
disordered in the 8PP structure, and the loop ends are depicted in red.
B, monomer of the ternary InhA⅐NADϩ⅐PT70 complex using the same colors
and orientation as in A. The substrate-binding loop is ordered in this structure
and covers the binding pocket (red cartoon). Secondary structure elements
for both molecules were assigned with STRIDE (34).
The resulting inhibitor, PT70, shows improved affinity for
InhA compared with the first generation compounds reported
previously (10, 11). Like the parent compound 5-hexyl-2-phe-
noxyphenol that lacks the methyl group (6PP; Fig. 1), PT70
ϩ
binds preferentially to the E-NAD product. However, the K1
value of 22 pM for PT70 is ϳ430-fold smaller than the KiЈ value
of 9.4 nM for 6PP (10), and thus introduction of the methyl
group has dramatically increased the affinity of the inhibitor for
the enzyme complex. More importantly, this inhibitor now also
displays slow onset inhibition, which is expected to be crucial
for its in vivo antibacterial activity. Assuming that kon for 6PP is
limited by the rate of encounter of enzyme and inhibitor (109
Additionally, the PT70 methyl group forms van der Waal’s
ϩ
contacts to the phosphate groups of the NAD cofactor, result-
ing in a 1 Å shift of the B-ring upward relative to its position in
8PP. In contrast, when triclosan is bound to InhA the B-ring is
tilted by ϳ25° toward Ile202 of the substrate-binding loop and
thereby interferes sterically with the loop residues, pushing the
loop away from the substrate cavity instead of keeping it in
place (Fig. 5).
M
Ϫ1 sϪ1), then the KiЈ value of 9.4 nM allows koff for 6PP to be
estimated at 9.4 sϪ1, giving a residence time of 0.1 s for 6PP on
the enzyme. This value is in stark contrast to the residence time
of 24 min for PT70, a 14,000-fold increase compared with 6PP.
ϩ
The crystal structures of the ternary InhA⅐NAD ⅐PT70 com-
DISCUSSION
plex reveal how the inhibitor is bound to the active site. The
We have used structure-based design to develop a slow onset observed hydrophobic interactions and the hydrogen bonding
inhibitor that directly targets InhA, the enoyl-ACP reductase of network of PT70 are similar to the observed interactions of
M. tuberculosis. Previous work resulted in the development of a previously described triclosan derivatives (10). In addition,
series of alkyl-diphenyl ethers that are nanomolar inhibitors of however, PT70 generates hydrophobic interactions to amino
InhA (10). The best inhibitor of this series, 8PP, is active against acids Ala198, Met199, Ile202, and Val203, which are part of the
drug-sensitive and drug-resistant strains of MTB. Although substrate-binding loop (residues 195–210 in InhA). This sub-
these inhibitors have high affinity for InhA, they are still rapid strate-binding loop is disordered in InhA structures in the pres-
reversible inhibitors. However, based on these encouraging ence of triclosan as well as in the structures with the improved
MAY 7, 2010•VOLUME 285•NUMBER 19
JOURNAL OF BIOLOGICAL CHEMISTRY 14335

Slow Onset Inhibition of InhA
ϩ
5-pentyl-2-phenoxyphenol and 8PP triclosan derivatives that,
The substrate-binding loop in the InhA⅐NAD ⅐PT70 struc-
despite their improved IC50 values, remain to be rapid reversi- ture adopts a different conformation compared with that
ble inhibitors (10). In contrast, triclosan is a slow onset inhibitor observed in other InhA complexes in which the substrate-bind-
of E. coli FabI, and the corresponding crystal structure shows ing loop is also ordered. The loop in the PT70-bound structures
that the substrate-binding loop is ordered (15). The substrate- is close to the substrate-binding pocket because of hydrophobic
binding loop is also ordered in the crystal structure of InhA interactions with the inhibitor, whereas the loops in the other
inhibited by the slow onset INH-NAD adduct (14). These structures are located farther from the pocket. Although the
observations prompted us to speculate that compounds with fatty acid in the substrate-bound structure (Protein Data Bank
the ability to cause loop ordering will be slow, tight binding code 1bvr (30)) does interact with residues Ala198, Ile202, and
inhibitors of InhA (10). Although loop ordering with respect Met199, movement of the loop in this case is thought to be
to INH-NAD binding remains elusive, the triclosan bound necessary to widen the binding pocket to accommodate the
E. coli FabI structure clearly reveals that the inhibitor forms long chain fatty acid. However, in the InhA complexes with the
hydrophobic interactions with the loop amino acids Ala196 triclosan derivative JPL (Protein Data Bank code 3fng (11)) and
and Ala197, which correspond to Ala198 and Met199 in InhA. the pyrrolidine carboxamide inhibited form (Protein Data Bank
Additionally, Rozwarski et al. (30) identified in the crystal code 2h7m (31)), movement of the loop away from the sub-
structure of InhA with a C16 fatty acyl substrate that hydro- strate-binding pocket could be due to the reduced number of
phobic amino acids of the loop are important for proper contacts to Ala198, Met199, and Ile202. Although a number of
substrate binding into the cavity. Interestingly, the last few contacts are present between enzyme and inhibitor in the PT70
carbon atoms of the fatty acid interact with the hydrophobic structure, a detailed analysis of the structural data suggests that
amino acids Ala198, Met199, and Ile202. A fatty acid shorter interactions with the side chain of Ile202, which is turned toward
than 16 carbons might not be accommodated correctly by the PT70 B-ring, is particularly important for orientation of the
the enzyme because of missing interactions with Ala198
Met199, and Ile202
,
substrate-binding loop close to the active site.
.
Although measurements of inhibitor residence time are not
These observations strongly suggest that the interactions normally incorporated into drug discovery programs, there is
with the three amino acids (Ala198, Met199, and Ile202) are growing evidence that residence time is a critical factor for in
important determinants for loop ordering. An inhibitor, like vivo drug activity (12, 13, 32, 33). In particular, slow onset inhib-
PT70, that is able to directly interact with these residues leads to itors will spend longer times bound to their targets compared
a defined loop structure. The ordered substrate-binding loop with rapid reversible inhibitors and will remain bound even
covers the entrance to the binding pocket and thereby locks the when free drug concentrations are low. Recent studies with the
inhibitor into the cavity and increases its residence time. It is FabI enzyme from F. tularensis highlight the importance of this
conceivable that the conformational change of the loop poses concept, where it was seen that residence time was a better
the slow step observed in the binding studies.
predictor of in vivo activity than the thermodynamic affinity of
It is important to note that Freundlich et al. (11) recently the inhibitor for the enzyme (13).
reported InhA structures inhibited by triclosan derivatives that
The new characteristics of PT70 facilitate the hydrophobic
have an ordered substrate-binding loop in two of the four struc- interactions to the important loop residues of InhA, resulting in
tures (Protein Data Bank codes 3fng and 3fnh). However, it was slow tight binding inhibition. Our work supports the prediction
not reported, whether the compounds are slow binding inhib- that slow onset inhibition of enoyl-ACP reductases is indeed
itors. The possibility that loop ordering is due to intermolecular coupled to loop ordering. Slow binding and improved residence
contacts in space group I4122, which has not been reported so time are expected to result in significant improvements in in
far for InhA structures, cannot be excluded. In this space group vivo antibacterial activity.
residues 197–205 of the substrate-binding loop are closely
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